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Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity
浸根接种法评估禾谷镰孢菌导致小麦根腐病的致病力   

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Molecular Plant Microbe Interactions
Dec 2015

Abstract

Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via root infection has been examined recently. F. graminearum has a unique infection strategy which comprises complex, specialized structures and processes. Root colonisation negatively affects plant development and leads to systemic plant invasion by tissue-adapted fungal strategies. The pathosystem wheat root - F. graminearum makes available an array of research areas, such as (i) the relatively unknown root interactions with a necrotrophic pathogen; (ii) genes and pathways contributing to (overall) Fusarium resistance; (iii) induced systemic (whole-plant) resistance; (iv) pathogenic strategies in a variety of host tissues; and (v) age-related changes in the single-genotype responses to seedling and adult plant (root/spike) infection. The presented Fusarium root rot bioassay allows for efficient infection of wheat roots, evaluation of disease severity and progress as well as statistical analysis of disease dynamics.

Keywords: Fusarium root rot (镰孢菌根腐病), Fusarium graminearum (禾谷镰孢菌), Root inoculation (根接种), Disease severity assessment (疾病严重度评估), qPCR-based diagnosis (基于qPCR的诊断), Repeated measures ANOVA (重复测量ANOVA), Host-pathogen interaction (宿主-病原体相互作用), Wheat (小麦)

Background

The Fusarium root rot (FRR) bioassay uses root-dipping for inoculation in combination with different measurements of disease severity parameter. This protocol is principally also applicable to investigations of other root-fungus interactions. The presented root-dip inoculation proved to be an effective and reliable method to investigate wheat root-Fusariuminteractions (phenotypically and histologically) and to screen wheat genotypes for their response to root infection (Wang et al., 2015). Using the described protocol, genetic, molecular, and metabolomic aspects of the FRR disease have meanwhile been examined with reliable results in terms of biological repetitions and consistent observations across different research approaches. This corresponds with observations made in a study on the Verticillium wilt disease, which characterised root-dipping as superior to the pot immersion or soil infestation method in terms of effectiveness and reliability (Trapero et al., 2013). Growing wheat seedlings in F. graminearum contaminated (root zone) soil led to FRR-genotype responses similar to root dip inoculation (Wang et al., 2015), but the infection conditions are comparatively less controlled in terms of root specificity and time of infection. This might be a restriction for investigations that require time-based analyses. The use of Petri dishes to germinate and inoculate roots via mycelial agar plugs is a method that has been applied to F. culmorum infection in wheat seedlings (Beccari et al., 2011). However, in comparison to root dip inoculation, this method is not applicable to adult plant root infection, as was done in our lab to study plant age-related effects on FRR disease progress and wheat responses.

In the described protocol, disease severity can be assessed by percentage reductions of diseased root biomass, root and shoot length as well as by rates of visible root necrosis. FRR significantly inhibits root biomass production of wheat seedlings and adult plants (Wang et al., 2015), which can be measured by quantitative real-time PCR (qPCR). Disease severity and progress in terms of fungal growth can be monitored by measuring the relative amount of F. graminearum DNA in the host tissue by qPCR. This also enables detection and monitoring of infection during or in case of symptom-free disease periods. The F. graminearum spread into the lower stem internode is a crucial event as it initiates the colonisation of upper stem internodes, leaves, further tillers and even spikes (Wang et al., 2015) and can be readily evaluated by appearance time and rate of visual necrosis. For the FRR disease progress over time, a good agreement was found between the quantified relative F. graminearum biomass in roots and the measured impacts on seedling growth or the rated visible symptoms (Wang et al., 2015). Briefly, seedlings with the lowest level of F. graminearum accumulation measured displayed relative minor root necrosis and reductions in root biomass and length, while relatively moderate and maximum levels of pathogen accumulation each led to correspondingly moderated and maximum disease impacts and symptoms. Finally, Fusarium resistance is quantitative or partial. Therefore, the combination of classical, subjective tools such as symptom rating with the sensitive, non-subjective qPCR diagnosis of pathogen and/or root biomass proved to be advantageous, in terms of an improved assessment of disease dynamics and genotype performances.

Materials and Reagents

  1. Parafilm
  2. Cheese cloth
  3. Fine sand (washed and sieved), obtained from construction or agricultural market, autoclaved at 120 °C for 30 min
  4. Aluminium foil
  5. Polystyrene disk (thickness 10 mm)
  6. F. graminearum isolate ‘IFA 65’ (University of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, Vienna, Austria)
  7. Synthetic nutrient agar medium ‘Spezieller Nährstoffarmer Agar (SNA)’ (Leslie and Summerell, 2006)
  8. Tween-20 (Carl Roth, catalog number: 9127 )
  9. MENNO Florades (MENNO CHEMIE Norderstedt)
  10. Sodium hypochlorite solution (Carl Roth, catalog number: 9062 )
  11. Wuxal Super (Manna, Düsseldorf)
  12. Liquid nitrogen
  13. Potato dextrose broth (PDB) (Sigma-Aldrich, catalog number: P6685 )
  14. FastStart Universal SYBR Green Master (Roche Molecular Systems, catalog number: 04913850001 or 04913914001 )
  15. Potassium dihydrogen phosphate (KH2PO4)
  16. Potassium nitrate (KNO3)
  17. Magnesium chloride heptahydrate (MgSO4·7H2O)
  18. Potassium chloride (KCl)
  19. Glucose
  20. Sucrose
  21. Agar
  22. Synthetic nutrient deficient agar (SNA) (see Recipes)

Equipment

  1. Climate chamber with 20 °C under cool-white and near-UV light illumination for preparation of fungal culture
  2. Haemocytometer
  3. Light microscope (Zeiss)
  4. Magnetic stirrer
  5. Climate chamber with a 16 h photoperiod of 22 °C/18 °C day/night and 60% humidity for plant cultivation
  6. Flat tray
  7. Rotary shaker
  8. Pot (7.5 x 7.5 x 8.0 cm)
  9. NanoDrop ND 1000 (Thermo Fisher Scientific, model: NanoDrop ND 1000 )
  10. ABI Step One Plus real-time PCR system (Applied Biosystems)

Software

  1. PSS 20 (IBM SPSS Statistics 20; IBM Corp., USA)

Procedure

  1. Preparation of F. graminearum macroconidia suspension
    1. Store macroconidia suspension at -80 °C in sterile, double-distilled water as stock solution.
    2. To prepare macroconidia suspension for inoculation, transfer 15 µl of stock solution on a synthetic nutrient deficient agar (SNA) and culture at 20 °C under cool-white and near-UV light illumination. Use Parafilm to seal the agar plates. Please also take into consideration Note 1 on fungal aggressiveness given at the end of protocol.
    3. The 9-day-old fungal colony is ready for harvest of macroconidia. SNA is a weak nutrient agar. Hence, colonies grow slow and produce relatively low amounts of (fluffy) mycelia, which at harvest time vary from white to light pink in colour (Figure 1). Details on the macroconidia morphology are given by Leslie and Summerell (2006).
    4. Wash the SNA agar medium with 4 ml 0.02% (v/v) Tween-20 solution (diluted in sterile, double-distilled water) and then pass through four layers of sterile cheese cloth. Determine the concentration of macroconidia suspension by using a haemocytometer and adjust the concentration to 5 x 104 macroconidia/ml with 0.02% (v/v) Tween-20.


      Figure 1. Fusarium graminearum colony on SNA plate at day of harvest

  2. Root-dip inoculation [at seedling stages]
    1. Keep seedlings free from contamination. Clean the chamber prior to cultivation and inoculation by using an antimicrobial disinfecting agent, according to the manufacturer’s specifications [in this case with MENNO Florades].
    2. Sterilize seeds in sodium hypochlorite (6%) for 40 min on a magnetic stirrer; then wash 10 times with sterile double-distilled water.
    3. Sow seeds in autoclaved sand and cultivate plants in a climate chamber with a 16 h photoperiod of 22 °C/18 °C day/night and 60% humidity until the first leaf is unfolded [Zadoks (Z) 11; Zadoks et al., 1974].
    4. Prior to inoculation, remove plants carefully from the sand.
    5. Pool every five seedlings by wrapping their hypocotyls and stems with aluminium foil. This will ensure that only roots come in contact with the fungal suspension and allows fixation of seedlings as shown in Figure 2. In addition, seedlings can be held in place by using a slot in a polystyrene plug. Plugs are cut out from a polystyrene disk (thickness 10 mm) to the size of flat tray. For each chamber a hole is punched (a conical point chisel is useful), large enough to let pass seedling roots.
    6. For inoculation, place pooled seedlings into a flat tray (divided into chambers) by submerging their roots in 5 ml of macroconidia suspension (Figure 2); then shake gently for 2 h on a rotary shaker (Figure 2). For non-inoculated control plants, perform mock inoculation with 0.02% (v/v) Tween-20 (diluted in sterile, double-distilled water).


      Figure 2. Root-dip inoculation of wheat seedlings with F. graminearum. Seedlings are placed into a small flat tray with 12 chambers each filled with 5 ml of macroconidia suspension. Hypocotyls and stems above the roots are wrapped in aluminium foil to avoid contact with the fungal suspension.

    7. After inoculation, transplant a single or at maximum five seedlings (not closely packed together) in a single pot (7.5 x 7.5 x 8.0 cm) with autoclaved sand. Cultivate seedlings in a climate chamber set at 20 °C under a 22 °C/18 °C day/night rhythm with approximately 60% relative humidity. Microscopic examinations have shown that F. graminearum actively penetrates the epidermal layer of roots by forming different specialised infection structures. To avoid disruption of the infection process, transplant the seedlings immediately after removal from fungal solution. No further treatment is needed. Add 100 ml of water after planting. The water should be poured slowly and carefully.
    8. During cultivation the watering (250 ml per pot) regime dependents on the present growth chamber (greenhouse) conditions. General rule: Sand has a limited water holding capacity. Hence, we suggest to water as soon as the sand surface is dry [in this case every 4 to 6 days]. Please also take into consideration the Notes 2 and 3 on plant cultivation and experimental setup given at the end of protocol.
    Note: The root-dip inoculation was likewise applied to adult plants at the early stem elongation stage (Z32-33). In contrast to seedling root inoculation, use a 1:2 (v/v) mixture of autoclaved sand and potting soil for cultivation before and after root-dip inoculation to ensure normal plant growth. After root inoculation, cultivate each a single plant per pot (11.5 x 11.5 x 12 cm); water as mentioned above for seedlings. Support the growth of adult plant with fertilizer to avoid undesirable additional stress. Fertilisation is carried out once every two weeks with 250 ml Wuxal Super according to the manufacturer’s specifications, or an equivalent fertiliser.

  3. Disease severity assessment
    1. Tissue sampling
      Collect (e.g., each 10) plants per sampling timepoint, treatment and genotype. Wash the roots under running water or immersed in water to avoid root injury. Roots should be washed until sand grains are removed from root surface. The parenchyma tissue of leaf sheath (wrapped around the young stem) is used by the pathogen to colonise lower stem. Hence, caution should be taken with washing, because diseased leaf sheath occasionally becomes very soft and drop gently.
    2. Disease impacts on plant development
      Measure the root and shoot length of inoculated and non-inoculated control plants. Calculate the relative reduction (percentage change) as the ratio between trait expression of diseased plants and trait expression of control plants:



    3. Visible disease symptoms
      Rate the symptoms on root and stem base by applying each browning and symptom extension scales (0 to 4) to single plants (Figure 3). The browning scale ranges from 0, symptomless; 1, slightly necrotic; 2, moderately necrotic; 3, severely necrotic; 4, completely necrotic; and the extension scale ranges from: 0, no lesions; 1, 1-24%; 2, 24-49%; 3, 50-75%; 4, > 75% discoloration of root or stem base. Calculate a genotype-timepoint-specific root and stem base symptom index (RSI and SbSI) by using the equation:
      RSI or SbSI = (E1 + E2 + … + En/n) + (B1 + B2 + … + Bn/n)
      Where,
      B and E each represent the parameter browning and extension scale,
      n represents the number of assessed individuals per genotype and treatment. Stem base is referred to the transition area between the root and the shoot system, located between sub-crown and first stem internode (Figure 3A).
      An overview on different degrees of diseased root phenotypes and necrosis symptoms is given by Wang et al. (2015). In this publication, Supplementary Figure S1 shows root phenotypes of four wheat genotypes, representing resistant and moderate to high susceptible responses. On the roots and stem bases of susceptible genotypes symptoms typically show a transition from initially local, light (amber) brown discolorations to more distributed dark brown lesions at later timepoints. Stem base necrosis typically covers about half of the stem base of initially few individuals and continued until nearly the entire stem base was affected for more or less all individual plants. Please also take into consideration the Note 4 on symptom assessment given at the end of protocol.
      Note: Create top view photos showing inoculated and non-inoculated plants side by side (Figure 3A). Such photos can, for example, be used for accurate measurement of browning extension by image analysis. 


      Figure 3. Wheat root phenotypes and necrosis symptoms. The example of FRR severity shows seedlings of the high-yielding cultivar Tobak. A. Seedlings at 14 days after root inoculation with Tween-20 (left) and F. graminearum (right). Inoculated seedlings show root and stem base necrosis as well as lowered root development. B. Close up of root and stem base symptoms visualised 21 days after root inoculation.

  4. DNA preparation for reference-gene-based qPCR quantification
    1. Select and pool root tissues of five seedlings per genotype/treatment/timepoint to be used for fungal and root biomass quantification. Freeze root samples in liquid nitrogen and store them at -80 °C until further use. Extract gDNA from root tissues according to the protocol given by Doyle and Doyle (1990). Dilute gDNA of all samples to 50 ng/μl as work solution.
    2. Preparation of gDNA as a plant standard in the qPCR quantification of root biomass. Collect leave samples from non-inoculated seedlings or young plants. Dilute gDNA serially with sterile double-distilled water (100, 50, 25, 6.25, 1.5625, 0.39 ng wheat DNA/μl) and keep DNA at -20 °C until use.
    3. For the isolation of gDNA from F. graminearum mycelium, inoculate 100 ml potato dextrose broth (PDB) with 2 ml of 2 x 105 macroconidia suspension and incubate on a rotary shaker at 28 °C for 3 to 5 days (depending on the mycelium growth). Harvest the mycelium by filtration: pass the PDB through four layers of sterile cheese cloth. Collect the mycelium, grind in liquid nitrogen and store it at -80 °C by using a mortar and pestle. Isolate the gDNA by applying the CTAB-based method described by Brandfass and Karlovsky (2008).
    4. Preparation of gDNA as a Fusarium standard in the qPCR quantification of fungal biomass. Dilute gDNA serially with sterile double-distilled water (50, 10, 5, 1, 0.5, 0.1 ng F. graminearum DNA/µl) and keep the DNA at -20 °C until use.
    Note: Pooling of root tissues is used due to the inability to obtain enough root material from a single seedling, especially at early timepoints after root infection and/or in the case of high susceptibility to FRR. To ensure consistency, pooling should be generally applied to all sampling timepoints (if several exist). In case adult plants are tested, pooling might be applied as a method of biological averaging.

  5. Reference-gene-based qPCR measurement of root and fungal biomass
    1. To amplify a F. graminearum specific fragment (Nicholson et al., 1998), use the primers Fg16N-F (ACAGATGACAAGATTCAGGCACA) and Fg16N-R (TTCTTTGACATCTGTTCAACCCA). To amplify wheat DNA (Ubiquitin gene, DQ086482) use the primers Ubi-F (CCCTGGAGGTGGAGTCATCTGA) and Ubi-R (GCGGCCATCCTCAAGCTGCTTA).
    2. Split PCR plate into two parts, each containing the wheat or fungal dilution series and eight root samples in three technical replicates. The amplification mix consists of 1 μl template DNA, 5 μl Roche FastStart Universal SYBR Green Master, 2 μl double-distilled water, and each 1 µl of forward and reverse primer (10 pmol/μl). Perform PCR with initial denaturation for 1.5 min at 95 °C; followed by 35 cycles with 30 sec at 94 °C, 45 sec at 64 °C, and 45 sec at 72 °C; and final elongation for 5 min at 72 °C.
    3. Analyse melting curves to ensure that only a single product was amplified. After amplifications, acquire the melting curves by heating the samples to 95 °C for 1 min, cooling to 55 °C for 1 min and then slowly increasing the temperature from 65 °C to 95 °C at the rate of 0.5 °C 10 sec-1, with continuous measurement of the fluorescence.
    4. Plot the CT values of each standard dilution series against the natural logarithm of the DNA concentration. Calculate the total amount of wheat DNA and fungal DNA with the gradient equation (f(x) = ax + b), according to the following formula (Livak and Schmittgen, 2001):



    5. Calculate the average amount of F. graminearum in wheat roots and the infection percentage of root samples (relative fungal biomass) by using the reference gene-based DNA quantification method and formula published by Brunner et al. (2009).
    6. Finally, calculate the relative fungal biomass as the ratio between fungal and wheat root DNA quantity:



      Where, total DNA is the sum of F. graminearum and wheat root DNA.
      Note: Calculate the average infection percentage as the average of the assayed technical and biological replicates.

  6. Fusarium root rot disease index (FDI) 
    The FDI incorporates all (or selected) severity parameter: relative fungal biomass; percentage change in root biomass, root length and shoot length; root and stem base symptom rating. Analogous to the area under the disease progress curve (AUDPC; Buerstmayr et al., 2000), the FDI can be used to rank genotypes for their overall performance under disease conditions (Wang et al., 2015) or as phenotypic parameter in genetic studies (Rutkoski et al., 2012).
    1. Transfer the measured values of each severity parameter into dimensionless susceptibility index (SI) values to allow normalisation and direct comparison between different types of value. Calculate SI values using the equation:
      SI = (XTx)/(Y)
      Where,
      X represents the measured value for each genotype and timepoint (Tx),
      Y the corresponding overall mean calculated over all assayed genotypes and timepoints.
    2. Calculate the respective FDI values for each genotype and timepoint as the sum of SI values.
      Note: In time-course studies FDI values can be calculated for each sampling timepoint.

Data analysis

Statistical analyses were performed by using the software SPSS 20 (IBM SPSS Statistics 20; IBM Corp., USA). The repeated measures ANOVA (rANOVA) was used to assess the time-course of FRR disease progression. In case the Mauchly’s test for sphericity violated the equality assumption, the Greenhouse-Geisser correction was applied to adjust the degrees of freedom appropriately. For multiple comparisons the Bonferroni adjustment (correction for Type I error) was applied. Two-way rANOVA was performed to test treatment and time effects on the traits root biomass, root length, and shoot length. Mixed rANOVA was performed to test for time effects (changes) in the disease progress in terms of fungal growth, reduction in root and shoot biomass of disease seedlings, and necrosis symptom development on roots/stem bases. Example tables for rANOVA results can be found in Wang et al., (2015).
Note: The mean profile plots (estimated marginal means) provided by the software SPSS 20 were applied to interpret significant time x treatment interactions. Examples for mean profile plots can be found in Wang et al. (2015). Tests of Within-Subjects Effects table provided by the software SPSS 20 gives three corrections for the significance of F-values: Sphericity assumed, Greenhouse-Geisser, Huynh-Feldt.

  1. Two-way rANOVA to test for significant treatment and treatment x time interactions in assessed severity traits [e.g., relative fungal biomass; root biomass and root length reduction; shoot length reduction]. This design includes the within-subjects factors ʻTimeʼ (independent factor with x sampling timepoints) and ʻTreatmentʼ (independent factor with FRR inoculated or non-inoculated control).
    Note: The traits relative fungal biomass, root and stem base symptom index are not examinable in non-inoculated control plants and thus, cannot be test in two-way design.
  2. The mixed rANOVA to test for significant time effects on the examined severity traits (dependent variables). This design includes the within-subjects factor ʻTimeʼ (as described for two-way rANOVA) and the between-subjects factor ʻGenotypeʼ (with x genotypes tested).
    Note: Here, no comparison between treatments is included and thus, also those traits can be tested which are only examinable in inoculated plants, while traits examinable in both inoculated or non-inoculated plants have to be analysed as calculated relative reduction values (see step C2).

Notes

  1. Variation in the disease severity can result from differences in the aggressiveness of fungal isolate. A reduced aggressiveness (infection rate) can result from long time storage on agar media. Therefore, we recommend to prepare fresh macroconidia suspension from stock solution (stored at -80 °C) prior to infection experiments.
  2. We preferred the cultivation of inoculated plants in a growth chamber over the greenhouse. In the growth chamber infection and disease progression were more reliable and comparable between independent experiments. For investigations on host-pathogen interactions almost stable environmental conditions ensure that the pathogen is the most prevalent factor that influences plant development. Indeed, for investigations on adult plants a growth chamber may provide not enough space.
  3. If available marker genotype(s) with well-characterised response(s) to root infection should be added to experiments to ensure that root inoculation was successful. For instance, a markedly and early visible symptom development on the stem base allows rapid estimation of infection.
  4. The necrosis symptom assessment might be challenged by certain factors that should be taken into account. In the seedling stages, necrosis rating on stem bases can be hampered by an early natural (non-pathogenic) browning of control stem bases, a phenomenon displayed by certain genotypes. Root necrosis is occasionally not clearly recognizable, although fungal growth and disease severity measurements demonstrate susceptibility to root infection. Whether this phenomenon results from experimental issues or pathogen behaviour is currently unanswered. Generally, we observed that rather dry conditions seem to support formation of root necrosis by the pathogen. Moreover, we suggest that F. graminearum is capable of evolving a symptom-free (rather endophytic) life style under certain conditions (Wang et al., 2015; Mudge et al., 2006). In this context, the disease spread within upper stem internodes is generally symptom-free and can only be monitored by using qPCR quantification of fungal biomass (Procedure E) or diagnostic PCR marker for F. graminearum. In the adult plant stages, visual symptom assessment is, to our experiences, impossible due to the plant age-related natural browning of roots and stem bases.

Recipes

  1. 1. Synthetic nutrient deficient agar (SNA)
    1 g KH2PO4
    1 g KNO3
    0.5 g MgSO4·7H2O
    0.5 g KCl
    0.2 g glucose
    0.2 g sucrose
    20 g agar
    Dissolve in 1 L double-distilled water; then sterilise by autoclaving at 120 °C for 20 min

Acknowledgments

We would like to thank Prof. Herrmann Buerstmayr and Prof. Marc Lemmens (University of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, Vienna, Austria) for kindly providing the F. graminearum inoculum. This work was partially supported by China Scholarship Council. This protocol was adapted or modified from the study: Wang, Q., Buxa, S. V., Furch, A., Friedt, W. and Gottwald, S. (2015). Insights into Triticum aestivum seedling root rot caused by Fusarium graminearum. Mol Plant Microbe In 28(12): 1288-1303. The research paper can be downloaded via ResearchGate accounts of Qing Wang and Sven Gottwald.

References

  1. Beccari, G., Covarelli, L. and Nicholson, P. (2011). Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum. Plant Pathol 60:671-684.
  2. Brandfass, C. and Karlovsky, P. (2008). Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error. Int J Mol Sci 9(11): 2306-2321.
  3. Brunner, K., Paris, M. P. K., Paolino, G., Burstmayr, H., Lemmens, M., Berthiller, F., Schuhmacher, R., Krska, R. and Mach, R. L. (2009). A reference-gene-based quantitative PCR method as a tool to determine Fusarium resistance in wheat. Anal Bioanal Chem 395(5): 1385-1394.
  4. Buerstmayr, H., Steiner, B., Lemmens, M. and Ruckenbauer, P. (2000). Resistance to Fusarium head blight in winter wheat: Heritability and trait associations. Crop Sci 40(4): 1012-1018.
  5. Doyle, J. J. and Doyle, J. L. (1990). A rapid total DNA preparation procedure for fresh plant tissue. Focus 12: 13-15.
  6. Leslie, J. F. and Summerell, B. A. (2006). The Fusarium laboratory manual. Blackwell Publishing 5: 388.
  7. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods (25):402-408.
  8. Mudge, A. M., Dill-Macky, R., Dong, Y., Gardiner, D. M., White, R. G. and Manners, J. M. (2006). A role for the mycotoxin deoxynivalenol in stem colonisation during crown rot disease of wheat caused by Fusarium graminearum and Fusarium pseudograminearum. Physiol Mol Plant P 69: 73-85.
  9. Nicholson, P., Simpson, D. R., Weston, G., Rezanoor, H. N., Lees, A. K., Parry, D. W. and Joyce, D. (1998). Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant P 53(1): 17-37.
  10. Rutkoski, J., Benson, J., Jia, Y., Brown-Guedira, G., Jannink, J. L. and Sorrells, M. (2012). Evaluation of genomic prediction methods for Fusarium head blight resistance in wheat. Plant Genome 5(2): 51-61.
  11. Trapero, C., Díez, C.M., Rallo, L., Barranco, D. and López-Escudero, F. J. (2013). Effective inoculation methods to screen for resistance to Verticillium wilt in olive. Sci Hortic 162: 252-259.
  12. Wang, Q., Buxa, S. V., Furch, A., Friedt, W. and Gottwald, S. (2015). Insights into Triticum aestivum seedling root rot caused by Fusarium graminearum. Mol Plant Microbe In 28(12): 1288-1303.
  13. Zadoks, J. C., Chang, T. T. and Konzak, C. F. (1974). A decimal code for the growth stages of cereals. Weed Res 14(6): 415-421.

简介

禾本科镰刀菌是小麦(小麦)和其他谷物中最常见和最有效的真菌病原体之一,已知会引起食品和饲料的破坏性产量损失和霉菌毒素污染。病原体主要被认为是镰刀菌枯萎病花粉病的典范,而最近已经检查了通过根系感染定植小麦植物的能力。 F。 graminearum 具有独特的感染策略,包括复杂的专门结构和过程。根系定殖对植物发育负面影响,并导致组织适应真菌策略的全身植物侵袭。小麦根系 - 小麦根。 graminearum 提供了一系列研究领域,例如(i)与坏死性病原体相对未知的根系相互作用; (ii)有助于(总体)镰刀菌抗性的基因和途径; (iii)诱导全身(全植株)抗性; (iv)各种宿主组织中的致病策略;和(v)对幼苗和成年植物(根/穗)感染的单基因型应答的年龄相关变化。提出的镰刀菌根腐病生物测定可以有效地感染小麦根,评估疾病的严重性和进展以及疾病动态的统计分析。

背景 根腐病(FRR)生物测定法使用根浸法进行接种,结合不同的疾病严重性参数测量。本协议主要适用于其他根真菌相互作用的研究。所提出的根浸接种被证明是一种有效和可靠的方法来研究小麦根 - 镰刀菌相互作用(表型和组织学)和筛选小麦基因型对根系感染的响应(Wang et al。 al 。,2015)。使用描述的方案,FRR疾病的遗传,分子和代谢组学方面同时经过不同研究方法的生物学重复和一致性观察结果的可靠结果进行了检查。这一点与在黄萎病的研究中所作的观察结果相对应,其特征在于在浸种或浸种方法方面的有效性和可靠性优于根浸渍(Trapero等人< /em>。,2013)。生长小麦幼苗。污染(根区)土壤导致类似于根浸种接种的FRR-基因型反应(Wang等人,2015),但感染条件在根系上较少受到控制特异性和感染时间。这可能是需要进行时间分析的调查的限制。使用培养皿通过菌丝体琼脂塞发芽并接种根,是已经应用于F的方法。小麦幼苗感染(Beccari等人,2011)。然而,与根浸种接种相比,这种方法不适用于成年植物根系感染,如我们实验室中所做的,研究与FRR疾病进展和小麦反应相关的植物年龄相关影响。
 在描述的方案中,疾病严重程度可以通过病害根生物量的减少百分比,根和芽长度以及可见根坏死率来评估。 FRR显着抑制小麦幼苗和成年植物的根生物量生产(Wang等,2015),可以通过定量实时PCR(qPCR)进行测定。可以通过测量F的相对量来监测疾病严重性和真菌生长方面的进展。通过qPCR在宿主组织中的禾谷镰刀菌DNA。这也可以在无症状疾病期间或在无症状疾病期间检测和监测感染。 F。传播到下茎节间的禾本科是一个关键的事件,因为它启动了上部茎节间,叶子,进一步分蘖甚至尖峰的定植(Wang等人,2015),并且可以通过外观时间和视觉坏死率容易地评估。对于随着时间的推移,FRR疾病的进展在量化的相对F之间发现了良好的一致性。根系中的禾本科生物量和对幼苗生长或额定可见症状的测量影响(Wang等人,2015)。简而言之,具有最低水平的幼苗。禾本科的积累测量显示相对较小的根坏死和根生物量和长度的减少,而相对中度和最高水平的病原体积累导致相应的缓和和最大的疾病影响和症状。最后,镰刀菌抗性是定量的或局部的。因此,在疾病动力学和基因型表现的改进评估方面,经典的主观工具如症状评估与敏感的,非主观的qPCR诊断病原体和/或根生物量的组合被证明是有利的。

关键字:镰孢菌根腐病, 禾谷镰孢菌, 根接种, 疾病严重度评估, 基于qPCR的诊断, 重复测量ANOVA, 宿主-病原体相互作用, 小麦

材料和试剂

  1. 石蜡膜
  2. 奶酪布
  3. 从建筑或农业市场获得的细砂(洗涤和筛分),在120℃下高压灭菌30分钟
  4. 铝箔
  5. 聚苯乙烯盘(厚度10毫米)
  6. F。禾本科分离'IFA 65'(自然资源与应用生命科学大学,农业生物技术系,奥地利维也纳)
  7. 合成营养琼脂培养基"SpeziellerNährstoffarmerAgar(SNA)"(Leslie and Summerell,2006)
  8. 吐温-20(Carl Roth,目录号:9127)
  9. MENNO花卉(MENNO CHEMIE Norderstedt)
  10. 次氯酸钠溶液(Carl Roth,目录号:9062)
  11. Wuxal超级(曼纳,杜塞尔多夫)
  12. 液氮
  13. 马铃薯葡萄糖肉汤(PDB)(Sigma-Aldrich,目录号:P6685)
  14. FastStart Universal SYBR Green Master(Roche Molecular Systems,目录号:04913850001或04913914001)
  15. 磷酸二氢钾(KH 2 PO 4)<>>
  16. 硝酸钾(KNO 3 )
  17. 七水合氯化镁(MgSO 4·7H 2 O)
  18. 氯化钾(KCl)
  19. 葡萄糖
  20. 蔗糖
  21. 琼脂
  22. 合成营养不足琼脂(SNA)(见食谱)

设备

  1. 气候室为20°C,在冷白光和近紫外光照射下,用于制备真菌培养物
  2. 血细胞计数器
  3. 光学显微镜(Zeiss)
  4. 磁力搅拌器
  5. 气候室,白天/晚上为22°C/18°C,植物栽培湿度为60%。
  6. 平托盘
  7. 旋转振动筛
  8. 锅(7.5 x 7.5 x 8.0 cm)
  9. NanoDrop ND 1000(Thermo Fisher Scientific,型号:NanoDrop ND 1000)
  10. ABI Step One Plus实时PCR系统(Applied Biosystems)

软件

  1. PSS 20(IBM SPSS Statistics 20; IBM Corp.,USA)

程序

  1. F的准备。 graminearum macroconidia暂停
    1. 将大分子悬浮液在-80℃下储存在无菌双蒸水中作为储备溶液
    2. 为了制备接种的大分子悬浮液,将15μl储备溶液转移到合成营养缺乏的琼脂(SNA)上,并在20℃下在冷白光和近紫外光照下进行培养。使用石蜡膜密封琼脂板。还请考虑议定书末尾给出的关于真菌侵略性的说明1。
    3. 9日龄的真菌殖民地已准备好收获大分子植物。 SNA是一种营养不良的琼脂。因此,殖民地生长缓慢并产生相对较少量的(蓬松)菌丝体,其收获时间从白色变成浅粉红色(图1)。关于大分子形态的细节由Leslie和Summerell(2006)给出。
    4. 用4ml 0.02%(v/v)Tween-20溶液(稀释在无菌双蒸水中)洗涤SNA琼脂培养基,然后通过四层无菌乳酪布。通过使用血细胞计数器确定大分子量悬浮液的浓度,并用0.02%(v/v)吐温-20将浓度调节至5×10 4大分子/ml。


      图1.收获日期SNA板上的禾谷镰刀菌菌落殖民地

  2. 根浸接种[苗期]
    1. 保持幼苗免受污染。根据制造商的规格(在这种情况下与MENNO Florades),使用抗菌消毒剂进行培养和接种之前,清洁室内。
    2. 用磁力搅拌器将种子在次氯酸钠(6%)中灭菌40分钟;然后用无菌双蒸水洗10次。
    3. 在高压灭菌的沙子中播种种子,并在气候室中培养植物,16小时光周期为22℃/18℃白天/60%湿度,直到第一叶展开为止[Zadoks(Z)11; Zadoks等人,1974]。
    4. 接种前,请小心地从沙子上取下植物。
    5. 通过用铝箔包裹它们的下胚轴和茎,来孵育每个5个幼苗。这将确保只有根与真菌悬浮液接触并允许固定幼苗,如图2所示。此外,通过使用聚苯乙烯插塞中的狭缝可以将幼苗保持在适当的位置。将塞子从聚苯乙烯盘(厚度10mm)切割成平坦托盘的尺寸。对于每个室,冲孔(圆锥形凿子是有用的),足够大以允许通过幼苗根。
    6. 对于接种,通过将其根部浸入5ml的大分子悬浮液中(图2)将合并的幼苗置于平坦的托盘(分为室)中。然后在旋转振荡器上轻轻摇动2小时(图2)。对于未接种的对照植物,用0.02%(v/v)吐温-20(稀释在无菌双蒸水中)进行模拟接种。


      图2.带有F的小麦幼苗的根浸接种。 gr um。。。>>>>。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。根部上的下胚轴和茎被包裹在铝箔中以避免与真菌悬浮液接触。

    7. 接种后,将单一或最多5只幼苗(不紧密包装在一起)移植到带有高压灭菌砂的单盆(7.5×7.5×8.0厘米)中。在温度为20°C,温度为22°C/18°C的气候室中培育幼苗约60%的相对湿度。显微镜检查显示,F。通过形成不同的专门的感染结构,禾谷镰刀菌积极地渗透根表皮层。为了避免破坏感染过程,在从真菌溶液中取出后立即移植幼苗。不需要进一步的治疗。种植后加入100毫升水。水应缓慢而小心地倒入水中。
    8. 在种植期间,浇水(每盆250毫升)依赖于目前的生长室(温室)条件。一般规则:砂具有有限的持水能力。因此,我们建议一旦砂面干燥[在这种情况下每4至6天]即可进行水分。请考虑议定书末尾给出的关于植物栽培和实验设置的注释2和3。
    注意:在早期茎伸长期(Z32-33),根浸接种同样应用于成年植物。与根苗接种相反,使用1:2(v/v)高压灭菌砂和盆栽土壤的混合物进行根浸前后的培养,以确保正常的植物生长。根根接种后,每盆培养单株植物(11.5×11.5×12cm);水如上所述为幼苗。支持成年植物与肥料的生长,以避免不必要的附加压力。施肥每两周进行一次,根据制造商的规格使用250毫升的Wuxal Super,或等效的肥料。

  3. 疾病严重性评估
    1. 组织采样
      每个采样时间点,处理和基因型收集(例如,每个10)植物。在流水下洗涤根或浸入水中以避免根部受伤。根部应清洗,直至从根表面除去砂粒。叶鞘的薄壁组织(缠绕在幼茎上)被病原体用于定植下茎。因此,洗涤时要小心,因为患病叶鞘偶尔会变得很软,轻轻地下降
    2. 疾病对植物发育的影响
      测量接种的和未接种的对照植物的根和苗长度。计算相对减少(百分比变化)为病态植物的性状表达与对照植物的性状表达之间的比例:



    3. 可见疾病症状
      通过将单个褐变和症状延伸量表(0到4)应用于单一植物来评估根和茎基部的症状(图3)。褐变量范围为0,无症状; 1,坏死坏死; 2,中度坏死; 3,严重坏死; 4,完全坏死;扩展范围为:0,无病变; 1,1-24%; 2,24-49%; 3,50-75%; 4,> 75%的根或茎基变色。通过使用以下方程计算基因型 - 时间点特异性根和茎基症状指数(RSI和SbSI):
      RSI或SbSI =(E1 + E2 + ... + En/n)+(B1 + B2 + ... + Bn/n)
      哪里,
      B和E各自表示参数褐变和扩展刻度,
      n表示每个基因型和治疗的评估个体数。茎基被称为位于子冠和第一茎节间之间的根和枝系统之间的过渡区域(图3A)。
      王氏等人给出了不同程度的病根表型和坏死症状的概述。 (2015)。在本出版物中,补充图S1显示了四种小麦基因型的根表型,代表抗性和中度至高度敏感反应。在敏感基因型的根和茎基础上,症状通常显示从最初的局部,轻(琥珀色)棕色变色到稍后时间点的分布较深的黑褐色病变的转变。茎基底坏死通常占最初几个人的茎基的约一半,并持续到几乎整个茎基受到或多或少的所有个体植物的影响。还请考虑议定书末尾给出的症状评估说明4。
      注意:创建并排显示接种和未接种植物的顶视图照片(图3A)。例如,这样的照片可以用于通过图像分析来精确测量褐变延伸。


      图3.小麦根表型和坏死症状 FRR严重程度的例子显示了高产栽培品种Tobak的幼苗。 A.用Tween-20(左)和F后接种14天后的幼苗。 graminearum (右)。接种的幼苗显示根和茎基坏死以及根系发育的降低。 B.根根接种后21天,根部和茎基症状的关闭可见。

  4. 基于参考基因的qPCR定量的DNA制备
    1. 选择和汇集每个基因型/处理/时间点的五棵幼苗的根组织,用于真菌和根生物量的定量。冷冻根液样品在液氮中,并将其储存在-80°C直到进一步使用。根据Doyle和Doyle(1990)给出的方案从根组织中提取gDNA。将所有样品的gDNA稀释为50 ng /μl作为工作溶液
    2. 在qPCR定量根生物量中制备gDNA作为植物标准品。从未接种的幼苗或幼苗收集样本。用无菌双蒸水(100,50,25,6.25,2.5625,0.39ng小麦DNA /μl)连续稀释gDNA,并将DNA保持在-20℃直到使用。
    3. 用于从F分离gDNA。 graminearum 菌丝体,用2ml 2×10 5大肠杆菌悬浮液接种100ml马铃薯葡萄糖肉汤(PDB),并在28℃的旋转振荡器上孵育3至5天(取决于对菌丝生长)。通过过滤收获菌丝体:将PDB通过四层无菌乳酪布。收集菌丝体,在液氮中研磨,并使用研钵和研杵储存在-80°C。通过应用Brandfass和Karlovsky(2008)描述的基于CTAB的方法分离gDNA。
    4. 在真菌生物量的qPCR定量中制备gDNA作为镰孢霉素标准品。用无菌双蒸水(50,10,5,1,0.5,0.1 ng禾谷物DNA /μl)连续稀释gDNA,并将DNA保持在-20°C直到使用。
    注意:由于不能从单个幼苗获得足够的根部材料,特别是在根感染后的早期时间点和/或在对FRR敏感性高的情况下,使用根组织的汇集。为了确保一致性,通常应将汇总应用于所有抽样时间点(如果有的话)。在成体植物进行测试的情况下,可以采用汇集作为生物平均方法。

  5. 基于参考基因的qPCR测量根和真菌生物量
    1. 放大F。特异性片段(Nicholson等人,1998),使用引物Fg16N-F(ACAGATGACAAGATTCAGGCACA)和Fg16N-R(TTCTTTGACATCTGTTCAACCCA)。为了扩增小麦DNA(泛素基因,DQ086482)使用引物Ubi-F(CCCTGGAGGTGGAGTCATCTGA)和Ubi-R(GCGGCCATCCTCAAGCTGCTTA)。
    2. 将PCR板分成两部分,每个包含小麦或真菌稀释系列,以及八个根样品三次技术重复。扩增混合物由1μl模板DNA,5μlRoche FastStart Universal SYBR Green Master,2μl双蒸水和各1μl正向和反向引物(10pmol /μl)组成。在95℃进行初始变性1.5分钟进行PCR;随后35次循环,94℃30秒,64℃45秒,72℃45秒;并在72℃下最终延伸5分钟。
    3. 分析熔解曲线,以确保只有一种产品被扩增。放大后,通过将样品加热至95℃1分钟,冷却至55℃1分钟,然后以0.5℃10秒的速度将温度从65℃缓慢升高至95℃,获得熔融曲线 -1 ,连续测量荧光。
    4. 按照DNA浓度的自然对数绘制每个标准稀释系列的C 值。用梯度方程式计算小麦DNA和真菌DNA的总量( x ) >),根据下列公式(Livak和Schmittgen,2001):



    5. 通过使用Brunner等人公布的参考基于DNA的DNA定量方法和公式计算小麦根中禾谷镰刀菌的平均含量和根样本的感染百分比(相对真菌生物量)(2009年) )。
    6. 最后计算真菌和真菌的生物量,作为真菌和小麦根DNA的比例数量:



      其中,DNA总数为总和。禾谷镰刀菌和小麦根DNA。
      注意:计算平均感染百分比作为测定的技术和生物重复的平均值。

  6. 镰孢根腐病指数(FDI)
    FDI包含所有(或选定)严重性参数:相对真菌生物量;根生物量,根长和芽长度百分比变化;根和茎基症状评分。类似于疾病进展曲线下的区域(AUDPC; Buerstmayr等人,2000),FDI可以用于在疾病条件下对基因型的整体表现进行排名(Wang等人,,2015)或遗传研究中的表型参数(Rutkoski等人,2012)。
    1. 将每个严重度参数的测量值转移到无量纲敏感指数(SI)值,以允许不同类型值之间的归一化和直接比较。使用以下公式计算SI值:
      SI =(XTx)/(Y)
      哪里,
      X表示每个基因型和时间点(Tx)的测量值,
      Y是在所有测定的基因型和时间点上计算的对应总平均值。
    2. 计算每个基因型和时间点的相应FDI值作为SI值的总和。
      注意:在课程研究中,可以为每个抽样时间点计算FDI值。

数据分析

统计分析使用SPSS 20软件(IBM SPSS Statistics 20; IBM Corp.,USA)进行。重复测量方差分析(rANOVA)用于评估FRR疾病进展的时间过程。如果Mauchly对球形度的测试违反了平等假设,则适用温室 - Geisser校正来适当调整自由度。对于多次比较,应用Bonferroni调整(修正I型误差)。进行双向rANOVA测试对根系生物量,根长和枝条长度的处理和时间效应。进行混合rANOVA测试疾病进展的时间效应(变化)方面,真菌生长,疾病幼苗的根和苗生物量的减少以及根/茎基上的坏死症状发展。关于rANOVA结果的示例表格可以在Wang等人,(2015)中找到。
注意:应用软件SPSS 20提供的平均分布图(估计边际平均值)来解释显着的时间x处理相互作用。平均轮廓图的示例可以在Wang等人中找到。 (2015)。受试者测试由软件SPSS 20提供的效应表给出了F值的意义的三个校正:假定的球形度,温室 - Geisser,Huynh-Feldt。

  1. 双向rANOVA用于在评估的严重程度性状(例如,相对真菌生物量)中测试显着的治疗和/或治疗x时间相互作用。根生物量和根长度减少;拍摄长度缩短]。该设计包括受试者内因素"时间"(具有x采样时间点的独立因子)和"治疗"(具有FRR接种或非接种对照的独立因子)。
    注意:相对真菌生物量,根系和茎基症状指标在非接种对照植物中不可检测,因此不能在双向设计中进行检验。
  2. 混合的rANOVA用于检验严重程度性状(因变量)的显着时间效应。该设计包括受试者内因子"时间"(如双向rANOVA所述)和受试者之间因子"基因型"(测试了x个基因型)。
    注意:在这里,没有包括治疗之间的比较,因此,也可以测试那些仅在接种的植物中检查的性状,而在接种或未接种的植物中可检测的性状必须作为计算的相对降低值(见步骤C2)。

笔记

  1. 疾病严重程度的变化可能是由真菌分离物侵袭性的差异引起的。琼脂培养基长时间储存可导致侵袭性降低(感染率)。因此,我们建议在感染实验前从储备溶液(储存于-80°C)制备新鲜的大分子悬浮液。
  2. 我们更喜欢在温室内的生长室中接种植物的培养。在生长室中,感染和疾病进展在独立实验之间更可靠和可比较。对于宿主 - 病原体相互作用的调查,几乎稳定的环境条件确保病原体是影响植物发育的最普遍的因素。事实上,对于成年植物的调查,生长室可能没有足够的空间
  3. 如果具有良好表征的根系感染响应的可用的标记基因型应加入实验中以确保根接种成功。例如,茎基上明显早期可见的症状发展可以快速估计感染。
  4. 坏死症状评估可能受到应考虑的某些因素的挑战。在幼苗阶段,通过对某些基因型显示的对照基因的早期天然(非致病性)褐变可以阻碍茎碱基上的坏死评价。根部坏死偶尔也不清楚,尽管真菌生长和疾病严重度测量显示对根感染的敏感性。这种现象是否来自实验问题或病原体的行为目前还没有得到答复。通常,我们观察到相当干燥的条件似乎支持病原体形成根坏死。此外,我们建议。 graminearum 能够在某些条件下发展无症状(相当内生的)生活方式(Wang等人,2015; Mudge等人,2006年) )。在这种情况下,在上部茎节间扩散的疾病通常是无症状的,并且只能通过使用真菌生物量(方法E)的qPCR定量或F的诊断性PCR标记来监测。 graminearum 。在成年植物阶段,根据我们的经验,视觉症状评估是不可能的,因为植物年龄相关的根和根茎的自然褐变。

食谱

  1. 合成营养不良琼脂(SNA)
    1g KH 2 PO 4
    1 g KNO <3>
    0.5g硫酸镁·7H 2 O 2·
    0.5克KCl
    0.2g葡萄糖
    0.2克蔗糖
    20克琼脂
    溶于1L双蒸水中;然后通过在120℃高压灭菌20分钟消毒

致谢

我们要感谢Herrmann Buerstmayr教授和Marc Lemmens教授(奥地利维也纳农业生物技术系自然资源与应用生命科学大学),慷慨提供了"F"。 graminearum 接种物。这项工作得到了中国奖学金委员会的部分支持。这个协议是从研究中改编或修改的:Wang,Q.,Buxa,SV,Furch,A.,Friedt,W.and Gottwald,S。(2015)。< a class ="ke-insertfile"href = "http://xueshu.baidu.com/s?wd=paperuri%3A%28dfd138d447af44290112e4baba5f3222%29&filter=sc_long_sign&tn=SE_xueshusource_2kduw22v&sc_vurl=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F26325125&ie=utf-8&sc_us = 17694268925617688386"target ="_ blank">对禾谷镰孢引起的幼苗根腐病的了解 28(12):1288-1303。研究论文可以通过Qing Wang和Sven Gottwald的ResearchGate账号下载。

参考文献

  1. Beccari,G.,Covarelli,L.和Nicholson,P。(2011)。感染过程和软小麦对由镰刀菌引起的根腐病和冠腐病的反应 植物Pathol 60:671-684。
  2. Brandfass,C.和Karlovsky,P.(2008)。用于镰刀菌镰刀菌的扩增的基于CTAB的DNA提取和实时PCR测定和/或F。 graminearum DNA在植物材料中具有减少的抽样误差。 Int J Mol Sci 9(11):2306-2321。
  3. Brunner,K.,Paris,MPK,Paolino,G.,Burstmayr,H.,Lemmens,M.,Berthiller,F.,Schuhmacher,R.,Krska,R.and Mach,RL(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19756538"target ="_ blank">基于参考基因的定量PCR方法作为确定>小镰孢菌抗性。分子生物学化学 395(5):1385-1394。
  4. Buerstmayr,H.,Steiner,B.,Lemmens,M。和Ruckenbauer,P.(2000)。抵抗镰刀菌冬小麦头部枯萎:遗传力和性状关联 作物科学 40 (4):1012-1018。
  5. Doyle,JJ和Doyle,JL(1990)。  快速总计新鲜植物组织的DNA制备程序。 焦点 12:13-15。
  6. Leslie,JF和Summerell,BA(2006)。< a class ="ke-insertfile"href ="http://as.wiley.com/WileyCDA/WileyTitle/productCd-0813819199.html"target ="_ blank" >镰刀菌实验手册 Blackwell Publishing 5:388.
  7. Livak,KJ和Schmittgen,TD(2001)。分析的相对基因表达数据使用实时定量PCR和2 -ΔΔCT方法。 方法(25):402-408。
  8. Mudge,AM,Dill-Macky,R.,Dong,Y.,Gardiner,DM,White,RG and Manners,JM(2006)。< a class ="ke-insertfile"href ="http: "sc ="_ blank">真菌毒素脱氧雪腐镰刀菌烯醇在禾本科镰刀霉和镰刀霉引起的小麦冠腐病期间的茎群定植中的作用ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium ium假性神经节。生理分子植物P 69:73-85。
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引用:Wang, Q. and Gottwald, S. (2017). Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity. Bio-protocol 7(6): e2189. DOI: 10.21769/BioProtoc.2189.
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